1. Field of the Invention
This invention is in the field of recombinant DNA technology and relates to fowlpox virus as a vector for foreign DNA.
2. Description of the Prior Art
Several viruses with DNA genomes have been used to carry and express genes from other viruses or other species. Viruses used in such a way are known as `vectors` and genes, other than their own, expressed in such a way are referred to as `foreign genes`. One of the primary requirements for a virus to be used as a vector in this manner is a suitable site for insertion of the foreign gene. If insertion of a gene into a site in the virus causes disruption of some function essential for growth, then such a site could not be used. Suitable sites are those at which an insertion does not disrupt any functions or those whose functions are not essential for viral growth and therefore can be disrupted with impunity. Such sites are known as `non-essential regions`. The phrase `non-essential` in this context means non-essential for growth under at least some conditions in which the virus can be grown in vitro and under at least some conditions in which it survives in vivo.
Examples of viruses which have been used as vectors by virtue of the fact that they contain non-essential regions are orthopoxviruses, adenoviruses and herpesviruses, although the regions used may be different in each case. Vaccinia virus (VV), which has been used as a vector, is an orthopoxvirus, a member of the pox virus family. Fowlpoxvirus (FPV), the subject of this patent application, is also a pox virus, but is a member of a different genus, the avipoxviruses. VV can be grown in tissue culture, and foreign genes can be inserted into the viral genome during this process. Several regions of VV have been found to be non-essential in vitro in more than one tissue-culture system. These include: large regions towards the left hand end, B. Moss et al., J. Virology 40, 387-395, (1981) who describe a mutant VV having a deletion 6.4 megaDaltons from the left-hand end; D. Panicali et al., J. Virology 37 1000-1010, 1981) who describe a mutant VV having a deletion starting 6.85 megaDaltons from the left-hand end; the thymidine kinase (TK) gene, D. Panicali and E. Paoletti, Proc. Natl. Acad. Sci. USA 79, 4927-4931 (1982); M. Mackett et al., J. Gen. Virol. 45, 683-701, (1982); and the vaccinia growth factor (VGF) gene, R. M. L. Buller it al., J. Virology 62, 866-974 (1988). Sites such as these might also be non-essential for growth in vivo. However in the case of both the TK and VGF gene, it has been found that although growth in tissue culture is unaffected or only slightly affected by insertion into the gene, growth and virulence in vivo are markedly affected, R. M. L. Buller el al., Nature 317, 813-815 (1985) and loc. cit. (1988), showing that these genes are not completely non-essential for growth of the virus in vivo. This in vivo attenuation may however be useful if it reduces unwanted pathogenic effects of the virus and accordingly growth in vivo with accompanying attenuation is a valid growth condition for the purpose of this invention.
Some sites are essential in some tissue culture systems and non-essential in others. For example there is a gene in VV which is essential for replication in human cells but which is non-essential in chicken embryo fibroblast cells, S. Gillard et al., Proc. Natl. Acad. Sci. USA 83, 5573-5577 (1986). Another gene in the related orthopoxvirus cowpox virus is essential for growth in chinese hamster ovary cells but not for growth in chicken embryo fibroblast cells, D. Spehner et al., J. Virology 62, 1297-1304 (1988). These examples show that differences in the tissue culture systems can affect which regions are non-essential.
The VV genome is far from being completely mapped and relatively little has been published about the FPV genome. It is known that FPV has a TK gene which has about 60% homology with the VV TK gene at the amino acid level , D. B. Boyle et al., Virology 156, 355-365 (1987). Like the VV TK gene, it serves as a non-essential region for homologous recombination under at least some conditions: see PCT Application WO 88/02022 (CSIRO). Using the E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene as a dominant selectable marker, in conjunction with the VV "7.5" promoter, the influenza haemagglutinin (HA) gene was inserted into a TK region of FPV and the FPV grown in chicken embryo skin cells. Expression of the HA gene was demonstrated by the binding of HA antibodies and labelled protein A to plaques of recombinant FPV.
The genome of FPV is known to have other similarities to VV in some regions. For example the DNA polymerase genes are closely related, having 42% homology at the amino acid level, M. M. Binns et al., Nucleic Acids Research 15, 6563-6573 (1987). On the other hand, F. M. Tomley, in a talk at the 6th Workshop on Poxvirus/Iridovirus, Cold Spring Harbor, N.Y., Sep. 24-28, 1986, described an attempted correlation of an 11.2 kb fragment of FPV DNA, located near one end of the genome, with VV DNA. While she reported 25% amino acid homology between a gene predicting a 48 kd polypeptide in FPV and a gene in VV coding for a 42 kd polypeptide, no other match of any significance was found. More detail of this work is given in F. M. Tomley et al., J. Gen. Virol. 69, 1025-1040 (1988).
F. M. Tomley et al, quoting M. Mackett and L. C. Archard, J. General Virology 45, 683-701 (1979), also state at page 1038 that the terminal portions of orthopoxvirus genomes are less well conserved than the central region and also say that the terminal regions, of around 30 to 35 kb in length, are thought to encode genes which determine specific virus characteristics such as host range, virulence, tissue tropism and cytopathogenicity and are thus less likely to be conserved.
The terminal region of VV is known to be of possible interest in relation to non-essential regions. Thus, M. E. Perkus et al., Virology 152, 285-297 (1986), have found that deletions in the VV genome occur in the presence of bromoxydeuridine, one of which extended from about nucleotides 2700 to 24100 from the left-hand end and that the deletion mutant virus is viable in tissue culture (cell type not clearly stated). Some information has been released about the very terminal region of FPV, in a poster by J. I. A. Campbell displayed at the International Poxvirus Workshop, Cold Spring Harbor, N.Y., Sep. 9-13, 1987. (The poster gives more information than does the abstract published in "Modern Approaches to New Vaccines including Prevention of AIDS", CSH 1987, page 45). Like VV and cowpox virus, FPV has terminal sequences which are inverted repeats of each other and are covalently cross-linked together. A part of the terminal inverted repeat (TIR) sequence was digested with BamHI and cloned. The BamHI fragment was described as having a length of 6.3 kb. The poster describes the general layout of the sequences, which from left to right comprise a short unique region, a set of tandemly repeated sequences and then a long unique region containing three possible open reading frames. The summary notes that this FPV terminal fragment shares the general pattern of nucleotide sequence with the terminal fragments of VV and cowpoxvirus, but also notes some marked differences. The sequence of part of the VV TIR, namely from approximately nucleotides 6800 to 9000 from the left-hand end, VV i s reported by S. Venkatesan et al., J. Virology 44, 637-646 (1982).
Large differences between the DNA sequence of VV and FPV are to be expected, since the FPV genome is estimated to be at least one third longer than that of VV. It seems likely, from the present knowledge as cited above, that many of the differences between the genomes of FPV and VV will be nearer to the termini than to the center of the genome. Thus, information about the terminal region of VV is of limited interest in relation to FPV.
It has been a problem to locate a well-defined non-essential region other than the TK gene, in fowlpox virus. Very recently (Apr. 20, 1989), in PCT application publication No. WO89/03429 (Health Research Inc.), FPV recombinants have been described, but the non-essential regions mentioned therein are not all well defined. The recombinants were generated merely by cleaving the FPV gene into fragments and trying the fragments with test constructs to see whether the foreign gene under test was expressed.
The VV HindIII-D fragment is one of the best characterised regions of the virus. The sequence of this 16060bp fragment has been determined, Niles et al., Virology 153, 96-112 (1986), and thirteen genes (D1-D13) have been identified.
Several temperature-sensitive mutants have been mapped to genes within HindIII-D and fine mapping has assigned these to D2, D3, D5, D6, D7, D11 and D13, Seto it al., Virology 160 110-119 (1987). These genes are therefore essential for virus replication. It is not known whether the remaining genes within the VV HindIII-D fragment are essential or non-essential for virus growth. Recent experiments have suggested (though not proved) that D8, which encodes a transmembrane protein, might be non-essential for propagation of vaccinia virus in tissue culture, Niles and Seto, Journal of Virology 62 3772-3778 (1988). In these experiments, a frameshift mutation was introduced into the carboxy-end of D8 which removed the carboxy-terminal 56 amino acids of D8. Virus containing this mutation had growth rates indistinguishable from those of wild type virus.
It was not predictable, however, whether the D8 gene would occur in fowlpox virus and if so whether it would be non-essential. Moreover, there was the problem of how to locate the D8 gene in the FPV genome which is relatively unmapped and is much larger than that of VV. Consequently, examination of the vaccinia Virus HindIII-D fragment did not indicate how to find further non-essential regions within FPV.